The presence of organelles, areas of biochemical specialization, separated from one another other by membranes, characterize eukaryotic cells. Such cellular organization necessitates elaborate mechanisms to effectively deliver the correct macromolecules to the correct locations, under the appropriate conditions, as well as mechanisms for the biogenesis, maintenance and inheritance of the organelles. Our focus is on the nucleus. Using genetic approaches available for our model eukaryotic organism, yeast Saccharomyces cerevisiae we previously identified Los1p. Los1p and its homologues (Xpo-t) function in tRNA nuclear egress. However, LOS1/Xpo-t is an unessential gene in all organisms that it has been possible to ablate its function, requiring that cells possess Los1p-independent tRNA nuclear export pathway(s). Aim 1 of the proposed work employs both candidate and genome-wide technologies to uncover the Los1p-independent tRNA nuclear export pathway(s). Until recently tRNA movement was regarded to be unidirectional from the nuclear site of synthesis to the cytosolic site of function. However, we discovered that the reverse also occurs. In fact, large pools of tRNA imported from cytoplasm quickly and reversibly reside in the nucleus under particular physiological conditions or in particular yeast mutants. This "retrograde tRNA nuclear import pathway" is likely a newly discovered level of gene expression for all eukaryotic organisms. Aim 2 seeks to understand the mechanisms that govern the retrograde pathway and its coordination with cellular metabolism. In Aim 3 we employ genome-wide approaches to learn how the complicated and dynamic nucleus is organized into domains that are not separated from each other by membranes. To date, we discovered necessary roles for N-acetylation and an integral membrane protein to appropriately tether our reporter to the inner nuclear membrane. We seek to identify other such gene products and to learn whether those already identified fulfill general roles in subnuclear organization. We anticipate that the information gleaned will have significant application in human disorders, such as cancers, Emery-Dreifuss muscular dystrophy and Hutchison-Gilford Progeria syndrome that result from inappropriate nucleus/cytosol dynamics and nuclear organization. [unreadable] [unreadable] [unreadable]